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No-cook process using Granular Starch Hydrolyzing Enzyme (GSHE) has been evaluated for Indian broken rice and pearl millet. One-factor-at-a-time optimization method was used to identify optimum concentration of GSHE enzyme, under the yeast fermentation conditions using broken rice and pearl millet as fermentation feedstock. To understand efficacy of GSHE enzyme to hydrolyze non-cooked broken rice and pearl millet, the chemical composition, fermentation efficiency and ethanol recovery were determined.
Keywords: Granular Starch Hydrolyzing Enzyme, Acid fungal protease, Yeast fermentation, Ethanol yield, Indian broken rice and pearl millet
Food and energy security have always been essential needs in various ways. This is due to their limited resources and their increasing demand by a growing human population [1, 2, 3]. At the same time demands of ethanol has been increasing since it is considered to be an alternative transportation energy source other than food consumption [4, 5]. Considerable attention has been given to ethanol production from various available sugar substrates such as molasses, sugar cane juice ; starchy materials like rice, millet, corn, sorghum, wheat, potato, cassava [3, 5, 7, 8, 9, 10]; as first generation ethanol and cellulosic materials as second generation ethanol . Pearl millet, broken rice and sorghum are the major starchy materials used by Indian distilleries not only for the production of potable alcohol  but also for the fuel purpose (http://www.icrisat.org/text/research/grep/homepage/sgmm/chapter12.pdf). Moreover, Indian distilleries use these raw materials based on their availability and cost since these are seasonal grains [12, 13].
The increasing price of crude oil and other fossil fuels have increased the interest in alternative fuel sources around the world [14, 15]. Fuel alcohol production from starch materials needs constant process improvement for meeting the economic payback by lowering the high price energy consumption and improvement in fermentation efficiency in order to be considered as a viable alternative to fossil fuel. At present, production costs for ethanol is INR 20 to 23 per liter from molasses based ethanol plant (1.0 INR = 0.0225683 USD), which is slightly higher than the Brazil using molasses (INR 14 to 16 per liter) . The Indian distilleries seek technological alternatives that would lower cost and provide higher margins in order to compete with gasoline and other fossil fuels. For the molasses based industry with 100KL per day capacity will require 450KWH power, 1620 to 1800 KL water per day for molasses dilution; and cooling water requirement will be 1080 KL per day. For a plant of such capacity, 2.0 to 2.3 MT of steam for 1.0 KL of ethanol production is required. In India, due to limited availability of molasses, molasses alone is not sufficient to meet the growing ethanol needs of the country, especially for use as a biofuel. Furthermore, the government of India is aggressively promoting the concept of blending petrol (gasoline) with ethanol to reduce dependence on petrol, and about 500 million liters of ethanol would be required every year, even if 10% ethanol is blended with gasoline (http://www.gujagro.org/agro-food-processing/molasses-base-alcohol-34.pdf). Thus, a number of distilleries have started converting their molasses based plants into cereal grain based ethanol production . However, ethanol production cost is INR 23 to 28 per liter in grain based technology compared to molasses based technology. The major factors for such higher production cost are considered to be raw materials, steam, electric power and cooling water required in enzymatic liquefaction; saccharification; fermentation; and distillation process. Moreover, depending upon the technology, raw material selection by industries, utility consumption will vary (http://ejournal.icrisat.org/mpii/v3i1/impi1.pdf) . Utility consumption involves energy, electricity, water cooling and heating. Water and energy (steam and cooling generates through water) are the most extensively used commodities in process industries. Water scarcity and environmental regulations on water wastes are a major concern nowadays. In particular, grain-based bio-ethanol plants are water and energy intensive [17.18].
Most biological processes apply the conversion of starchy materials of the grain or cereals into glucose and their conversion into ethanol consists of three different steps, starch liquefaction (80 to 125oC), saccharification (55 to 65oC) and fermentation (32 to 35oC) of sugar to ethanol . Advanced development, has further reduced one enzymatic process step of separate saccharification (55 to 65oC) since the energy /resource/utility of availability is the major concern to the industry as these factors directly impact on production cost . The improved biological process of starch materials conversion is liquefaction and SSF (Simultaneous Saccharification and Fermentation) a process in which saccharifying enzyme further hydrolyzes the liquefied starch into fermentable sugars at yeast fermentation conditions and simultaneously fermentation of sugars to ethanol . However, the SSF has not impacted more on energy reduction since liquefaction of starchy materials takes place at higher temperature ranging from 80 to 125oC [1, 20, 21] requiring enormous amount of steam in liquefaction and also a efficient cooling water system to bring down the temperature from 80-125oC to 32-35oC for SSF process [19, 22]. Granular starch hydrolyzing enzyme (GSHE) developed by Genencor, a Danisco Division was used to hydrolyze no-cook starch directly to fermentable sugars under yeast fermentation conditions without using steam, and moreover improve the conversion efficiency of starch to ethanol due to less loss of sugars without high temperature cooking process and less biomass produced due to the less stress of yeast.
Thus, the objective of present study was to determine efficiency of GSHE enzyme under the yeast fermentation conductions using Indian broken rice and pearl millet as fermentation feedstock.
2. Materials and Methods
2.1 Enzymes, reagent and chemicals
Granular starch hydrolyzing enzyme is enzymes cocktails containing fungal alpha amylase and a glucoamylase that work synergistically to hydrolyze granular starch to glucose (STARGENâ„¢ 002, activity minimum 570 GAU/g, one Glucoamylase Unit [GAU] is the amount of enzyme that will liberate one gram of reducing sugars calculated as glucose per hour from soluble starch substrate under the conditions of the assay, www.genencor.com); FERMGENâ„¢ (acid fungal protease, activity minimum 1000 SAPU/g, the activity of FERMGENâ„¢ protease is expressed in Spectrophotometric Acid Protease Units [SAPU]. One SAPU is the amount of enzyme activity that liberates one micromole of tyrosine per minute from a casein substrate under conditions of the assay, www.genencor.com); SPEZYMEâ„¢ FRED (alpha-amylase, activity minimum 17,400 LU/g, one Liquefon Unit [LU] is the measure of the digestion time required to produce a color change with iodine solution, indicating a definite stage of dextrinization of starch substrate under specified conditions, www.genencor.com); and Optidexâ„¢ L-400 (glucoamylase, activity minimum 350 GAU/g, one Glucoamylase Unit [GAU] is the amount of enzyme that will liberate one gram of reducing sugars calculated as glucose from a soluble starch substrate per hour under the specified conditions of the assay, www.genencor.com) were obtained from Genencor a Danisco Division. Active Dry Yeast from AB Mauri India Pvt. Ltd (MIDC -415 722, India) and urea from Merck (ML7M573074; 60848605001730) were purchased. Industrial grade Indian broken rice and pearl millet grains were purchased from local market.
2.2 Milling of Indian broken rice and pearl millet
Whole grain of Indian broken rice and pearl millet were milled using laboratory milling grinder (Milcent, Anand, Gujarat-India) at a setting of B. A sieve analysis showed that 90% of flour of Indian broken rice and pearl millet had a particle size was passing through U.S. standard 40 mesh-sieves.
2.3 Chemical composition of Indian broken rice and pearl millet
Oil, tannin, total free P2O5, crude fibers and fat (lipid) contents in broken rice and pearl millet were analyzed as described in AOAC 18th EDN.:2006.
The moistures in milled flour of Indian broken rice and pearl millet were analyzed by using moisture balance (MOC-120H, Shimadzu).
2.5 Soluble glucose and fructose content
Soluble glucose and fructose in Indian broken rice and pearl millet flour were extracted in water. For that, 1.0gm of Indian broken rice/pearl millet flour (dry basis) was dissolved in 99ml of water and mixed for 1hr at ambient temperature. Sample was then analyzed by HPLC (Agilent Isocratic system 1200, USA) on an Aminex Column HPX-87H (catalogue number 1250140, Bio-Rad) at 60oC with a mobile phase of 0.01N sulfuric acid at a flow rate of 0.7ml/min. A standard containing glucose (0.5%) and fructose (0.5%) was used to identify and quantify the products.
% Soluble Glucose = (% Glucose/100) X [100/ (grain weight, (g) (% Dry Solid/100))] X (100) 
% Soluble Fructose = (% Fructose/100) X [100/ (grain weight, (g) (% Dry Solid/100))] X (100) 
2.6 Starch content
For analyzing the starch content in Indian broken rice and pearl millet grain, the grains were milled in such a way that 10% of particles retained onto U.S. standard 40sieve. The grain flour was hydrolyzed using enzymatic method where alpha-amylase, SPEZYMEâ„¢ FRED and glucoamylase, OPTIDEXâ„¢ L-400 were used for liquefaction and saccharification process, respectively. The end product glucose was further analyzed by HPLC (Agilent Isocratic system 1200, USA) on an Aminex Column HPX-87H (catalogue number 1250140, Bio-Rad) at 60oC with a mobile phase of 0.01N sulfuric acid at a flow rate of 0.7ml/min. A standard containing glucose (0.5%) was used to identify and quantify the product. Total glucose was calculated by using equation number three. The starch content was calculated by using equation four.
% Total Glucose = (% Glucose/100) X [100/ (grain weight, (g) (% Dry Solid/100))] X (100) 
% Starch = (% Total Glucose in grain sample - % Soluble Glucose in grain sample) X 0.9
(Enzyme treated sample) (Water Extracted Sample)
2.7 Protein content
The protein content in Indian broken rice and pearl millet feedstock was estimated by the Kjeldahl's Method (IS 7219:1973(Reaff.2005))
2.8 Optimization of GSHE enzyme concentration for ethanol production under the yeast fermentation conditions using Indian broken rice and pearl millet as fermentation feedstock
A 25% DS (dry solid) slurry of Indian broken rice and pearl millet flour as fermentation feedstock was prepared in 1 liter flask separately by adding the RO water. The pH of the slurry of Indian broken rice and pearl millet flour was adjusted to 4.5 using 6N H2SO4. A one-factor-at-a-time optimization method was used to identify the optimum concentration of Granular Starch Hydrolyzing Enzyme (GSHE), STARGENTM002, under yeast fermentation condition using Indian broken rice and pearl millet as fermentation feedstock. The STARGENTM 002 (GSHE) concentration of 1.5, 2.0, 2.5 and 3.0 kg per MT of grain was used for both the grains. At the same time, FERMGENâ„¢ (proteases), 0.2 kg per MT of grain; urea, 400ppm; and active dry yeast, 0.25% were added into 25% DS slurry of Indian broken rice and pearl millet. Flask was then covered with sterile plug and initial weight of flask was recorded prior to incubating at 32±2oC onto the rotary shaker at 300rpm. The flask weight (gm) and medium pH was measured at every 24hr intervals of fermentation process to calculate the ethanol production (%, w/w) based on weight loss or CO2 released by using following equations.
1MT of grain to Ethanol (L) =
1000/ [Grain weight, gm X (Initial Surry weight, gm - 24hr intervals slurry weight, gm) X 46] /44/0.789 
Ethanol production (%, w/w) based on CO2 released = (Total grain used, gm X 1MT grain to ethanol, Liter)/ (24hr intervals slurry weight X 0.789) 
2.9 Ethanol yield, residual starch and sugar analysis
After 72hr of fermentation, the slurry was distilled at 80oC by using Soxhlet's apparatus (Ambassader; B.P. Industries, Delhi-India). The distilled ethanol (% v/v at 20oC) was measured by using alcometer. At the same time, residual sugar in fermented slurry was estimated by the Fehling's method and residual starch was determined using enzymatic method where alpha-amylase, SPEZYME â„¢ FRED and glucoamylase, OPTIDEXâ„¢ L-400 were used for liquefaction and saccharification process, respectively. The end product was also estimated by the Fehling's method. 1% glucose was used as standard to determine the Fehling Factor for further quantification of residual sugar and starch.
2.10. Ethanol recovery and Fermentation Efficiency:
After laboratory distillation of the fermented slurry, ethanol recovery (liter per MT of grain), 95.5% ethanol recovery (liter per MT of grain) and fermentation efficiency (%) were further calculated by using following equation number 7, 8 and 9, respectively.
Ethanol Recovery (Liter per MT of grain) =
(Total volume of slurry, ml X Ethanol %, v/v at 20oC) /Total grain, gm 
95.5% ethanol recovery (Liter per MT of grain) =
(Ethanol recovery, lit per MT of grain X 0.809)/0.789 
Fermentation efficiency (%) =
(Total slurry, gm X Ethanol %, v/v at 20oC X 100)/ (Total grain, gm X % starch X 1.11 X 0.646)
All the experiments were done in triplicates and the values presented were the means of three independent determinations.
3.1 Composition of Indian broken rice and pearl millet
Composition content (%) of 10.03, moisture; 68.45, starch; 0.34, soluble glucose; 0.08, soluble fructose; 9.38, protein; 1.76, fat (lipid); 0.72, P2O5; 2.51, crude fibers; 0.12, tannin; 3.43, oil; and 3.23 others, which include (non-starch-polysaccharide, minerals, ash content, etc) were found in Indian broken rice whilst 10.45, moisture; 60.00, starch; 0.63, soluble glucose; 0.45, soluble fructose; 8.34, protein; 5.90, fat (lipid); 1.37, P2O5; 4.18, crude fibers; 0.28, tannin; 5.48 oil; and 2.91, others were observed in Indian pearl millet.
3.2 Optimization of GSHE enzyme concentration for ethanol production based on CO2 released
One-factor-at-a-time optimization method was used to identify optimum concentration of GSHE enzyme, under the yeast fermentation conditions using Indian broken rice and pearl millet separately as fermentation feedstock. The ethanol production (% w/w at 20oC) was calculated based on weight loss or CO2 released. Increasing concentration of GSHE resulted in increased ethanol production (% w/w at 20oC) was observed in Indian broken rice (Fig 1A) and pear millet (Fig 1B) fermentation feedstock. Furthermore, an optimum ethanol production was observed at concentration of 2.5 kg per MT of grain when Indian broken rice (Fig 1A) and pearl millet (Fig 1B) was used as fermentation feedstock. But further increasing the concentration of GSHE enzyme (under yeast fermentation conditions) did not have much impacted in enhancing the ethanol production like how it was observed at GSHE dosage of 2.0 and 2.5 kg per MT of grains (Fig 1A and B). Henceforth, 2.5kg per MT of grain concentration was considered an optimum dosage for an industrial scale ethanol production through this technology.
3.3 pH profile of fermentation medium processed at various GSHE concentrations under yeast fermentation conditions
pH of the fermentation medium was also monitored in each concentration of GSHE enzyme under yeast fermentation conditions using Indian broken rice (Fig 2A) and pearl millet (Fig 2B) as fermentation feedstock. The pH fermentation medium was found to be decreased from 4.5 to average 3.69 in each experimental study of Indian broken rice and pear millet feedstock.
3.4 Ethanol yield after distillation
Fermentation slurry was distilled after 72hr cycle. Distilled ethanol yield was estimated by using alcometer and reading (%, v/v) was calibrated at 20oC. Fermentation containing 25% dry solid of Indian broken rice having 68.45% starch resulted in 11.23±0.08, 11.53±0.10, 11.93±0.06 and 12.09±0.07% v/v at 20oC ethanol yield was observed in 72hr of yeast fermentation when GSHE enzyme was used at concentration of 1.5, 2.0, 2.5 and 3.0 Kg/MT of grain, respectively along with 0.2 Kg of FERMGN per MT of grain whilst in Indian pearl millet of 25% dry solid having 60% starch with the same enzymes concentrations and experimental conditions resulted in 9.60±0.09, 10.03±0.05, 10.46±0.06 and 10.48±0.04% v/v at 20oC ethanol yield was observed, respectively. Furthermore, based on these values, ethanol recovery was also calculated to liter per MT of the grain; and also to 95.5% ethanol liter per MT of grain (Table 1), considering fact that this technology is not limited only to use for fuel ethanol production but also can be used for portable purposes.
3.5 Fermentation efficiency, residual sugar and starch content
In each feedstock, increasing concentration of GSHE resulted in increased fermentation efficiency was observed (Table 2). Residual sugar was not detected in all experiments. The residual starch was observed in very minimal amount (Table 2).
The chemical and nutritional quality of fermentation feedstock of broken rice and pearl millet varies considerably from one geological place to another, and this may be attributed to genetic factors; environmental influences; fertilizer treatments; degree of milling; and storage conditions. It has been reported that these factors also impact in ethanol yield . Thus, it was necessary to understand substrate composition prior to the ethanol production through non-cook process. Therefore, this study was conducted. It has been reported that at higher temperature cooking in conventional process, the chemical components of grains get inactivated or may be toxic to the yeast, which further interferes with ethanol yield [1, 24] http://www.afripro.org.uk/papers/Paper08Hamaker.pdf. Moreover, It has also been reported that following no-cook process can impact their value in DDGS (Distilled Dry Grains Solids) quality or alternatively, these chemical components can be further converted into monomers by using enzymatic process to create additional nutritional value to the yeast growth [1, 25]. With this objective, acid fungal protease (FERMGENTM) along with various dosage of GSHE enzyme (STARGENTM 002) was used in initial stage of GSHE process under yeast fermentation conditions. This acid fungal protease hydrolyzes the proteins present in these grains in form of amino acids, peptides, and free amino nitrogen (FAN) essential for yeast growth. Furthermore, it has been reported that protease plays a key role not only in hydrolyzing the protein matrices in the kernel that binds the various fractions, which releases "hard" to hydrolyze starch; but also in faster ethanol rates; and higher ethanol yield for grain based substrates as compared to those without protease . While using acid fungal protease (FERMGENTM) along with various concentration of GSHE enzyme (STARGENTM 002) for Indian broken rice and pearl millet feedstock separately under yeast fermentation conditions, optimum ethanol production was observed at 60hr of fermentation cycle.
Running yeast fermentation at pH 4.0 to 4.5 is a routine practice to control contaminating bacteria in an industrial scale process . The decreasing in pH during the yeast fermentation is due to CO2 formation . Moreover, decreasing in pH is also may be due to accumulation of organic free nitrogen formed by FERMGENTM (acid fungal protease) during GSHE process. These assimilated nitrogen uptake by yeast produces H+ ions resulted in slight decreasing in pH of fermentation medium was observed. This kind of phenomenon has also been demonstrated by Castrillo et al.  that the assimilation of one ammonium mole by yeasts leads to the release of one H+ mole in solution. In further support of this study, it further has been shown that between 40 and 160 hr of fermentation of grape must, the ethanol concentration increases in the medium, which can explain pH drop during this period .
In comparison to both the grains feedstock, the ethanol production was obviously higher in broken rice than pearl millet that because of the level of starch content (in broken rice it was observed to be 68.45% whilst in pearl millet it was 60%). Furthermore, these research studies were conducted for both the feedstock to emphasize that this no-cook process technology is not limited to Indian broken rice feedstock only but can also work efficiently for Indian pearl millet offering an economic viability of the ethanol industry in India. Sharma et al.  have reported 9.1 % v/v ethanol yield in GSHE treated 100% Amioca starch having 15% dry solids, under yeast fermentations conditions. Hicks et al.  reported the VHG (Very High Gravity) fermentation of a hulled variety of barley (starch content of 59.9%), which yielded an ethanol concentration of 14.87±0.06% in which the pretreatment step was followed prior to GSHE process. Duan and Bade  have reported that by using GSHE process for Chinese rice under yeast fermentation conditions resulted in 430 to 470 liter ethanol recovery per MT of Chinese rice whilst by following conventional process with same substrate resulted in 380 to 400 liter absolute ethanol recovery per MT of Chinese rice. Duan and Shetty  have reported that use of phytase along with GSHE enzyme for sorghum under yeast fermentation conditions, resulted in 380-400 liter absolute ethanol recovery per MT of sorghum. It has been further reported that addition of phytase along with GSHE enzyme under yeast fermentation conditions has further improved the quality of DDGS for animal feed application . However, there is not a single report available on Indian broken rice and pearl millet for the GSHE cold process with or without any pretreatment in ethanol production.
In comparison to no-cook process, it has been reported that in conventional process having higher liquefaction's temperatures, theoretically, 100 g of starch is expected to produce 56.7 g of ethanol as a maximum yield, assuming that starch is completely converted into glucose. However, in practice only 81 to 90% of fermentation efficiency was observed in conventional process . Wu et al.  have followed three steps conventional process, in ethanol production from US pearl millet having 65.30% starch and 25% dry solid concentration; involved liquefaction at 95oC for 45min followed by 80oC for 30min; saccharification at 60oC for 30min, and yeast fermentation that resulted in ~ 11% v/v at 20oC ethanol yield with fermentation efficiency of 90% and residual starch 3.45%. Zhan et al.  have followed same conventional process for US sorghum having 68.8% starch and 25% dry solid concentration resulted in 10.72% v/v ethanol yield with 85.93% fermentation efficiency.
This drop in fermentation efficiency in conventional process is due to the loss of some fermentable sugars as a result of a heat-catalyzed Maillard reaction between amino acids and reducing sugars during jet-cooking . Furthermore, it has been observed that presence of soluble glucose and fructose in broken rice and pearl millet would be a ready for the yeast utilization in no-cook process whilst in cooked process (conventional process), at higher temperature due to Maillard reaction, these free sugars become inactive ; hence it would not be utilized by yeast in fermentation. Apart from this, in terms of process length, typical conventional process follows either three steps (liquefaction, saccharification and fermentation) or two steps processes (liquefaction and SSF, simultaneous saccharification and fermentation), whilst in no-cook process, all these biological process steps take place in a single step without providing any steam to cook the starchy materials. [32, 36, 37]. It is also know that ethanol fermentation based on "Granular starch hydrolysis" was associated with better recovery of value-added products in comparison with the traditional jet-cooking fermentation / conventional process [1, 3, 38].
It has also been reported that biomass [yeast] (1.95 kg per 100 kg starch) produced in the no-cook process was less than conventional process (3.88 kg per 100 kg starch), which partially explain the increase of conversion efficiency, as more sugars were used for ethanol instead of yeast growth .
Present investigation revealed the potential of no-cook process by using GSHE enzyme (STRAGENTM 002) along with acid protease enzyme (FERMGENTM) for Indian broken rice and pearl millet feedstock in ethanol production under yeast fermentation conditions. Furthermore, this no-cook process replaces conventional process in ethanol production; having benefits of steam savings and less capital investment / process simplification by reducing unit operations (single step process), This way, it can save operational cost and at same time it is an environmental friendly process and improve fermentation efficiency.
We sincerely acknowledge Dr. Jay Shetty, Genencor®, A Danisco Division for reviewing this research article.